Therapeutic delivery of proteins and cells, such as stem cells is currently accomplished by either systemic administration or bolus injection at the injury site. In both cases, only a relatively small percentage of active remains at the injury site and the bulk of the active end up in other organs like the liver and the lungs. An approach in which the actives are retained at the injury site would allow for lower dosage, less side effects and potentially higher activity.
Current approaches for cell and protein delivery include microspheres, physisorption to matrices like foams and covalent attachment to such matrices. Often these approaches are compatible with delivery through small gauge needles required for delicate tissues like the brain and heart. Hydrogels have the benefit that they are deformable and in some cases can be formed in situ. However, the predominant mechanisms of hydrogel formation are through photo-crosslinking or radical cross-linking, both of which cause harm to proteins and cells. A few approaches have been reported that are cell and protein compatible. Enzymatically cross-linked gels and gels formed by cross-linking through Michael addition have been reported, however the rate of formation is such that these gels need to be pre-formed ex situ and then introduced in situ.
Self-assembling peptide systems have the benefit that when designed properly, they can be in a liquid state ex situ and rapidly transform into a gel state when in contact with body fluid in situ. Since the gelation mechanism is based on non-covalent interactions, no harm to proteins and cells is expected. A system based on peptides with alternating hydrophobic and charged residues has been reported that self-assemble due to beta sheet type hydrogen-bonding interactions between the peptides. The potential drawback of these systems is that it relies on a delicate balance in charge which limits the scope of accessible sequences. Hydrogels based on Fmoc-protected diphenylalanine are prepared by injecting a solution of the peptide in hexafluoroisopropanol, an undesirable solvent and there may also be concerns about the toxicity of the Fmoc protecting group upon degradation of the peptide. A variant was reported in which Fmoc-protected phenylalanine is coupled to phenylalanine by an enzyme. Potential drawbacks are potential immune response to the enzyme and a slower reaction rate. Systems based on beta hairpin peptides have been reported that are introduced by shear-thinning through a cannula after which they re-form. Finally hydrogels based on beta-sheet forming peptide sequences and hydrogels based on coiled coils of alpha-helical peptides have been reported as well. Finally, there is an extensive body of work from around linear peptide amphiphiles which are peptides that on either the C- or N-terminus are functionalized with an alkyl tail. The advantage of these systems is that the additional energy gain of the hydrophobic collapse of the tails stabilizes the system. Linear molecules (single head, single C6-C22 tail) of which the peptide head group can be branched and bola-amphiphiles (head-tail-head arrangement) are described. A drawback of this system is that in general supra-physiological concentrations of divalent ions or substantial pH change is required to induce gelation. In addition, these gels do not display shear-thin behavior.
There are four major classes of amphiphiles: linear amphiphiles (single head, single tail), bola-amphiphiles (head-tail-head), gemini amphiphiles (two head groups, two tails) and dicephalic amphiphiles (two head groups, one tail). It has been shown that these classes exhibit different aggregation behavior compared to one another. An advantage of gemini and dicephalic amphiphiles is that they are ‘pre-polymerized’ and therefore form stable aggregates at lower critical aggregation concentrations. Gemini amphiphiles based on multiple positively charged peptides have been reported for use as gene transfection agents. Three dicephalic amphiphile systems are described hereafter. The first reported system comprises an amphiphile with a diphosphate head group that forms rectangular micellar aggregates. The two other systems have ammonium or saccharide head groups and form micelles.
In light of the drawbacks of the hydrogels described above, there is still a need for alternative hydrogels for bioactive delivery. In particular, there is a need for in situ gelling self-assembly hydrogels that are easily delivered through a small gauge needle, gel upon delivery under physiological conditions, and do not have an adverse effect on the bioactive.